Cerebral Cortex August 2008;18:1758--1770

doi:10.1093/cercor/bhm199

Advance Access publication November 21, 2007

Cux-2 Controls the Proliferation ofNeuronal Intermediate Precursors of theCortical Subventricular Zone

Beatriz Cubelos1,2, Alvaro Sebastian-Serrano1, Seonhee Kim3,

Carmen Moreno-Ortiz1, Juan Miguel Redondo2, Christopher

A. Walsh3 and Marta Nieto1

1Centro Nacional de Biotecnologıa, CSIC, Darwin 3, Campus de

Cantoblanco, Madrid 28049, Spain, 2Fundacion Centro Nacional

de Investigaciones Cardiovasculares, Melchor Fenandez

Almagro 3, Madrid 28029, Spain and 3Division of Genetics,

Children’s Hospital Boston and Howard Hughes Medical

Institute, Beth Israel Deaconess Medical Center, Harvard

Medical School, Boston, MA 02115, USA

Whereas neurons of the lower layers (VI--V) of the cerebral cortexare first born from dividing precursors at the ventricular zone, upperlayer neurons (II--IV) subsequently arise from divisions of in-termediate neuronal precursors at the subventricular zone (SVZ).Little is known about mechanisms that control the proliferation ofSVZ neuronal precursors. We herein report that the restrictedexpression of the homeodomain transcription factor Cux-2 in theSVZ regulates the proliferation of intermediate neuronal precursorsand the number of upper layer neurons. In Cux-2-deficient mice(Cux-22/2), there is excessive number of upper layer neurons andselective expansion of SVZ neuronal precursors. Double-labelingexperiments demonstrate that Cux-22/2 upper layer precursorsreenter the cell cycle in a higher frequency than wild-typeprecursors. Overexpression studies indicate that Cux-2 controlscell cycle exit in a cell-autonomous manner. Analysis of Cux-12/2;Cux-22/2 double mutant revealed that Cux-2 controls SVZproliferation independently of Cux-1, demonstrating that this isa unique function of Cux-2, not redundant with Cux-1 activities. Ourresults point to Cux-2 as a key element in the control of theproliferation rates of the SVZ precursors and the number of uppercortical neurons, without altering the number of deep corticallayers.

Keywords: cerebral cortex, Cut, Cux-2, late-born neurons, progenitor,transcription factor

Introduction

The vertebrate cortex is organized into distinct layers, with the

neurons within each layer sharing similar functions, morphol-

ogy, and birthdates. An important advance in the understanding

of brain development is that we now know that the sequential

birth of the distinct cortical layers is achieved via the

generation of an intermediate neuronal precursor for the

upper layers. Whereas neurons of the lower layers arise first

from asymmetric divisions of the radial glial cells in the

ventricular zone (VZ) (Malatesta et al. 2000; Noctor et al.

2001), neurons of the upper cortical layers are born from

intermediate neuronal precursors dividing symmetrically in the

subventricular zone (SVZ) (Haubensak et al. 2004; Miyata et al.

2004; Nieto et al. 2004; Noctor et al. 2004; Zimmer et al. 2004;

Englund et al. 2005; Wu et al. 2005; Cappello et al. 2006). This

2-step mechanism of development has been proposed to

confer an evolutionary advantage by enabling both the

expansion of cortical layers and the appearance of new

neuronal types (Englund et al. 2005; Gotz and Huttner 2005;

Hill and Walsh 2005; Kriegstein et al. 2006).

Indeed, the evolution of the cerebral cortex extended the

cortical surface area and increased the number of neuronal

layers and neuronal circuits. Furthermore, the number of

distinguishable upper layers increases from mice to humans

(reviewed in Marin Padilla 2001) so that the circuit diagram

differs between primates and other mammals (Hill and Walsh

2005).

Remarkably, this increment in the number of superficial

layers of the cortex that occurs during evolution correlates

with an expansion of the SVZ during embryonic development.

This might be indeed attributable to an increase in the number

of intermediate neuronal precursors. Furthermore, the selec-

tive expansion of the upper layers in the outer part of the

cortical gyri and of their precursors at the SVZ has recently

been proposed to contribute to the formation of the gyri

typical of the cerebral cortex of carnivores and primates

(Kriegstein et al. 2006).

Any account of the evolution and development of the

cerebral cortex therefore requires an understanding of the

molecular basis regulating the proliferation of SVZ precursors

and differentiation of the upper layer neurons. Several

molecules have been reported to control precursor cell

proliferation (Takahashi et al. 1995; Chenn and Walsh 2002;

Nowakowski et al. 2002), and the sequential activation of

transcription factors governs the acquisition of specific laminar

fates (Englund et al. 2005; Guillemot et al. 2006). However,

little is known about factors involved in the selective pro-

liferation and differentiation of SVZ precursors and upper layer

neurons (Calegari et al. 2005; Guillemot et al. 2006; Glickstein

et al. 2007). Genes selectively expressed in the neuronal

precursor population of the SVZ during embryonic develop-

ment, such as SVET, Tbr-2, Nex/Math-2, and Cux-2 (Tarabykin

et al. 2001; Nieto et al. 2004; Zimmer et al. 2004; Englund et al.

2005; Wu et al. 2005), may participate in these processes.

Remarkably, 2 of these genes, Cux-2 and SVET, are also

expressed in the upper layer neurons.

Cux-2 encodes a vertebrate homolog of the Drosophila

transcription factor Cut (Quaggin et al. 1996). In the peripheral

nervous system, Cut determines the neuronal fate of external

sensory organ precursor cells (Bodmer et al. 1987; Blochlinger

et al. 1990) and the dendrite morphology of their neuronal

progeny (Grueber et al. 2003). Upper layer neurons express

Cux-2 and a second Cut homolog, Cux-1, which does not show

the same highly SVZ restricted expression during development

(Nieto et al. 2004). The function of mammalian Cux genes in

the nervous system is unknown. The overlapping expression

patterns of Cux-1 and Cux-2 suggest redundant functions for

these genes in neural cells and accordingly Cux-1-deficient

mice (Cux-1–/–) show no specific phenotype related to the

development of the nervous system (Luong et al. 2002).

However, the restricted expression pattern of Cux-2 in the

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SVZ suggests specific functions for this transcription factor,

possibly in the generation of neurons of the upper cortical

layers. To address these issues, we generated a Cux-2-deficient

mouse strain by targeted gene deletion and investigated the

development of the cerebral cortex in these animals.

Our analysis of Cux-2–/– mice demonstrates that Cux-2

negatively regulates the proliferation of precursors of the

upper layer neurons in the SVZ, which reenter the cell cycle at

a higher frequency in the Cux-2–/– mice. Thus, our results

point to Cux-2 as a gene, the expression of which limits the

proliferation of SVZ intermediate precursor. In this context,

mechanisms modulating Cux-2 expression and functions

appear to be candidate for playing important roles in the

expansion of the cerebral cortex that occurs during evolution.

Experimental Procedures

Animals, Construction of a Mouse Cell Line ConditionallyTargeting Cux-2/Cutl2, and Generation of Cux-2–/– Mice

All animal procedures were approved by the Centro Nacional de

Biotecnologıa Animal Care and Use Committee, in compliance

with National and European Legislation. Cux-2 gene encodes 4

DNA-binding domains: 3 Cut repeats and 1 homeodomain. A

targeting construct was designed to conditionally eliminate

exons 22 and 23, which encode the third Cut repeat and part of

the homeodomain (exons 22 and 23) near the C terminus of the

Cux-2 protein, and to create a premature stop codon 3# after thedeletion. The targeting vector was constructed using a 1.9-kb

DNA fragment that contains regions located 1.4 kb 5# and 0.5 kb

3# of exon 21. The middle arm, containing exons 22 and 23, is

a 1.7-kb polymerase chain reaction (PCR) product. The long arm

is a 7.5-kb NdeI genomic fragment from a mouse lambda library.

A total of 10 lg of targeting vector was linearized with NotI and

then transfected into J1-129 embryonic stem cells by electro-

poration. After selection in G418, surviving colonies were

expanded and analyzed by Southern blot to identify clones that

had undergone homologous recombination. Correctly, targeted

ES cell lines were microinjected into C57BL/6J blastocysts.

Resulting chimeric mice were crossed with C57BL6 mice to

obtain Cux-2+/loxP mice. Mice carrying the conditional allele

(Cux2loxP) were mated with mice expressing CRE recombi-

nase under the human beta-actin promoter (Tg (ACTB-

CRE)2Mrt deleter mice; Jackson Laboratories, Bar Harbor, ME)

on a Swiss Webster background, to obtain mice giving germ line

transmission of the floxed null allele. Heterozygous (het) mice

for this allele (hereafter Cux-2+/–) were crossed with wild-type

(WT) mice to segregate from the beta-actin transgene. Cux-2+/–animals were mated to obtain Cux-2 homozygous mutant mice

(Cux-2–/–). Previous studies with antibodies that recognize

different regions of the Cux-2 protein have found 2 isoforms of

Cux-2 protein in a human cell line. Both isoforms are recognized

by an antibody against the N-terminal part of the protein (anti-

Cux-2 antibody 356) encoded by the undeleted part of the

targeted Cux-2 gene. Anti-Cux-2 antibody 356 shows immuno-

reactivity with mouse Cux-2 (Gingras et al. 2005). Western blot

analysis of the expression of Cux-2 protein in Cux-2–/– mice

using antibody 356 reveals 2 bands in adult cerebral cortex

homogenates (Supplementary Fig. 1). No bands were detected

on brain homogenates from adult Cux-2–/– animals (Supple-

mentary Fig. 1). Immunohistochemistry (IHC) studies using

antibody 356 shows expression of Cux-2 protein in the WT

brains but not in Cux-2–/– animals. These experiments

demonstrate the complete absence of Cux-2 protein and lack

of Cux-2 truncated mutated forms of the protein in the Cux-2–/–

animals. Cux-1–/– mice have been described previously (Luong

et al. 2002) and were obtained from A. J. van Wijnen (University

of Massachusetts Medical School, MA, USA). Animals were

maintained on a C57BL6: Swiss Webster background. Morning

of the day of the appearance of the vaginal plug was defined as

embryonic day (E) 0.5.

Southern Blot and PCR

Genomic DNA was obtained from ES cell clones or tail biopsies

and digested with KpnI. Southern blot was performed using

ExpressHyb hybridization solution (BD Biosciences, Mountain

View, CA) according to the manufacturer’s protocol. A 1-kb

cDNA probe corresponding to exon 24 was used to screen for

positive ES cell clones and to genotype mice carrying the loxP

and null alleles. Mice were screened by Southern blot and the

PCR. Primers for the detection of the null floxed allele were 5#-AAGGGCGGTGATTACAGAGA-3# and 5#-GCCTGCTGTGGTA-GACAGGT-3#; primers for the WT allele were 5#-TCAGCA-CATGGTGTCTGGAT-3#and 5#-CCTACTTTCTGCCTGCTTG-3#.PCR was carried out over 35 cycles of 94 �C for 1 min, 60 �Cfor 1 min, and 72 �C for 1 min in 2.5 mM Mg2Cl and 5 mM

Betaine.

Antibodies, IHC, and Histology

Mice were perfused transcardially with 0.1 M phosphate-

buffered saline (PBS; pH 7.4) followed by cold 4% para-

formaldehyde in PBS. The perfused brains were removed and

postfixed in 4% paraformaldehyde at 4 �C overnight. Fixed

brains were cryoprotected in 30% sucrose in PBS and sectioned

on a cryostat to produce either 10--20 lM cryosections on

Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh,

PA) or 50--100 lM floating cryosections. Sections were blocked

for 1 h at room temperature (r.t.) with 5% horse serum in PBS

containing 0.5% Triton-X 100 (blocking solution) and then

incubated for 1 h at r.t. or overnight at 4 �C with primary

antibodies diluted in blocking solution. Fluorescent-tagged

secondary antibodies (in PBS, 5% horse serum) were applied for

1 h at r.t., and sections were counterstained with Hoechst

33342 (Molecular Probes, Eugene, OR) and mounted in Aqua-

polymount mounting medium (Poly-Labo, Strasbourg, France).

Peroxidase/diaminobenzidine IHC staining was performed as

described (Cubelos et al. 2005). Briefly, after incubation with

primary antibody, sections were incubated with biotinylated

donkey anti-rabbit IgG (Sigma, St Louis, MO) for 1 h. Sections

were then washed 3 times in PBS, incubated with streptavidin-

biotinylated horseradish peroxidase complex, washed, and

incubated with 0.1 mg/ml H2O2 and 0.5 mg/mL diaminobenzi-

dine in PBS. Sections were mounted in glycerol--gelatin.

The following primary antibodies were used at the dilutions

indicated: rabbit polyclonal anti-Cux-1 (clone M222) (1:10) and

anti-Brn-1 (1:50) (Santa Cruz Biotechnologies Inc., St Cruz,

CA), rabbit polyclonal antiphosphohistone H3 (pH3) (1:500)

(Upstate, Spartanburg, SC), rabbit anticleaved caspase-3 (1:500)

(Cell Signaling Tech Inc., Danuers, MA), rabbit anti Ki67

(Novocastra, Newcastle on Tyne, UK), rabbit polyclonal anti-

Cux-2 (antibody 356, a gift from Dr Alex Nepveu of McGill

University Health Centre, Canada), rat anti-bromodeoxiuridine

(BrdU) (1:50) (BD Biosciences), and rabbit anti-Tbr-2 antibody

Cerebral Cortex August 2008, V 18 N 8 1759

(1:500) (Chemicon Inc., Hampshire, UK). Goat anti-rabbit and

goat anti-rabbit and anti-mouse secondary antibodies, conju-

gated to Alexa 488 and 594, respectively (Molecular Probes),

were applied at 1:500. Before staining for Cux-1, Cux-2, Ki67,

BrdU, clorodeoxiuridine (CldU), iododeoxiuridine (IdU), or

pH3, sections were boiled for 30 min in antigen retrieval

solution (Vector Laboratories, Burlingame, CA). This was

followed in the case of staining for BrdU, CldU, IdU, and pH3

by 30-min treatment with 2 N HCl.

Staining for CldU and IdU was performed as described (Aten

et al. 1994). Briefly, slides were incubated overnight at 4 �Cwith a mouse monoclonal anti-BrdU antibody (1:50; BD

Biosciences; cross-reactivity with IdU), then washed in Tris

buffer, pH 8.0, containing 0.5 M NaCl and 1% Tween 20 for

20 min at r.t. to eliminate background and possible cross-reactivity

with CldU, and incubated overnight at 4 �C with rat mono-

clonal anti-BrdU antibodies (1:250; Abcam, Cambridge, UK;

cross-reactivity with CldU). Secondary anti-mouse conjugated

to Alexa 495--conjugated and anti-rat Alexa 598--conjugated

antibodies (1:500; both from Molecular Probes) were applied

for 2 h at r.t. The presence of single positive neurons for IdU

and CldU in the stained cortical sections demonstrated the

specificity of the antibodies and the staining.

BrdU, CldU, and IdU Injections and Cell Counting

All BrdU, CldU, and IdU quantification analysis show results

obtained from the primary sensory cortex (primary somato-

sensory cortex barrel field [S1BF]. Interaural 2.34-2.22, Bregma

1.46-1.58 according to the mouse atlas of Paxinos and Franklin

1997). Similar results were observed in the primary motor

cortex and other regions of the sensory cortex (M1 and pri-

mary somatosensory cortex forelimb region [S1FL], according

to the mouse atlas of Paxinos and Franklin). BrdU, IdU, and

CldU were administered intraperitoneally at 5 mg/mL in PBS. A

single CldU injection was given 10 h after IdU injection at

E14.5. Anatomically matched sections were selected from each

mouse at each stage after BrdU injection (n = 3 Cux-2+/?control mice and n = 3 Cux-2–/– mice), and BrdU was detected

by IHC. The total cortical thickness was subdivided into 10 bins

of equal area, and the number of cells in which at least half of

the nucleus was BrdU positive was counted in each bin.

Identical results were obtained from independent analyses by

2 investigators.

Cortical Layer Thickness and Neuronal Density

For neuronal cell density and cortical thickness, serial sections

were cut from brains of control and Cux-2–/– animal (n = 3).

Then sections were Nissl stained, matched, and photographed.

Measurements and cell counts were performed on the primary

sensory cortex (S1BF, Interaural 2.34-2.22, Bregma 1.46-1.58

according to the mouse atlas of Paxinos and Franklin 1997).

Similar results of increased layer thickness were observed in

S1FL and M1 (Paxinos and Franklin 1997). Analysis was

performed blind by 2 investigators in independent analyses.

Cell density was calculated using the optical fractionator

method (Sterio 1984). Optical dissector was given by a

preexisting microscope grid (x, y, z = 61, 61, 10 lm). For cor-

tical thickness, 4--5 measurements were taken of each brain

region and mean values and standard deviations obtained. Statis-

tical significance was calculated using the 2-tailed Student’s

t-test.

Confocal Microscopy and Imaging

Confocal microscopy was performed with a Radiance 2100

(Bio-Rad, Hercules, CA) Laser Scanning System on a Zeiss

Axiovert 200 microscope. For fluorescence excitation, an argon

ion laser (488 nm), a Krypton--Neon laser (543 nm), and a red

diode (637 nm) were employed. The filter combinations used

for detection of Alexa 488 and Alexa 594 were a 560 DCLPXR

beam splitter and HQ 515/30 emission filter and a 650 DCLPXR

beam splitter with HQ 590/70, respectively. Sequential images

were taken with LaserSharp v5.0 software (Bio-Rad) and

analyzed using LaserPix v.4 image software (Bio-Rad).

Neurosphere Culture, Nucleofection, and Constructs

Dorsal telencephalons of E14.5 WT embryos were dissected

and cells dissociated as described (Nieto et al. 2004).

Dissociated single cells were cultured in DMEM-F12 supple-

mented with N2, fibroblast growth factor (10 ng/mL), and

epidermal growth factor (10 ng/mL) (Gibco, Invitrogen,

Carlsbad, CA). Neurosphere cultures were passaged every 3

days by mechanical dissociation. Dissociated cells were

cotransfected using the nucleofector technic (Amaxa Biosys-

tems, Gaithersburg, MD) with 2.5 lg each of DNA constructs

containing Cux-2 and green fluorescence protein (GFP) cDNAs

under the cytomegalovirus enhancer, chicken b-actin pro-

moter, and rabbit b-globin poly(A) signal (CAG) (cytomegalo-

virus [CMV] and beta-actin) promoter or the empty vector and

GFP construct. Neurosphere formation was monitored after

10 days in culture.

Results

Generation of Cux-2–/– Mice

To investigate the functions of Cux-2 in the development of

the nervous system, we generated mice carrying a null allele of

Cux-2 and analyzed the resulting brain phenotype. The

mutation was designed to remove exons 22--23 that encode

the third Cut repeat and the homeodomain near the C-terminal

end of the protein. Het Cux-2+/– mice were obtained by

breeding mice carrying a Cux-2 conditional allele (Cux-2+/loxP) with CRE deleter mice (see Experimental Procedure and

Fig. 1a--c). Homozygous Cux-2 mutant mice (Cux-2–/–) were

obtained by crossing het Cux-2+/– animals. Cux-2–/– mice

were born at the expected Mendelian ratios and showed

overall normal development and growth (not shown). To

confirm that the mutation of the Cux-2 C terminus effectively

removed expression of Cux-2 protein in mutant mice, postnatal

brain sections of WT and Cux-2–/– mice were stained with

a Cux-2 antiserum against the N-terminal part of the protein

that recognizes the 2 described isoforms of Cux-2 protein

but does not recognize Cux-1 (Gingras et al. 2005). This

demonstrated the complete absence of Cux-2 immunoreactiv-

ity in all brain regions of Cux-2–/– animals, including the upper

layer neurons and neurons of the ventrolateral and piriform

cortex (Fig. 1d, left panels). Western blot experiments

confirmed the complete absence of Cux-2 protein in Cux-2–/–

cortical homogenates (Supplementary Fig. 1). In contrast,

staining with a Cux-1-specific antiserum showed that the Cux-1

protein expression was indistinguishable between Cux-2–/–,

Cux-2+/–, and WT littermates (Fig. 1d and not shown). Thus,

loss of Cux-2 protein expression does not affect the expression

1760 Cux-2 Controls Upper Layer Precursor Proliferation d Cubelos et al.

of Cux-1. Also unaffected were the expression patterns of the

layer-specific transcription factors, Brn-1 (II-V), Foxp-1 (III--V),

Id-2 (II, III, V, and VI), and Tbr-1 (VI) (Bulfone et al. 1995;

Rubenstein et al. 1999; Hevner et al. 2001; McEvilly et al. 2002;

Sugitani et al. 2002; Ferland et al. 2003) (Fig. 1e). These results

thus show that in mice completely lacking Cux-2 protein

expression, upper and lower layer neurons each acquire their

correct laminar position and correctly expressed the early

layer-specific markers that we analyzed.

Cell Density and Total Neuronal Numbers Are Increasedin the Upper Layers of the Cux-2–/– Cerebral Cortex

Visual analysis indicated that adult Cux-2–/– brains were

moderately but consistently bigger in volume than WT brains

Figure 1. Targeting strategy for Cux-2 null mutation. (a) A Neo-TK cassette flanked by loxP sites (black arrowheads) was inserted downstream of a 1.5-kb fragment containingCux-2 exons 22 and 23. A third loxP site was introduced 1-kb upstream of exon 22. Cux-2þ/loxP het mice were bred with mice expressing CRE recombinase under the humanbeta-actin promoter to obtain mice bearing the floxed null allele (Cux-2 allele) (b). (c) Southern analysis of the null mutation in the germ line. The Southern blot probe used isa 500-bp fragment of exon 24 (panels a and b). (d) Expression of Cux-2 and Cux-1 protein in the WT and Cux-2�/� cortex. Expression of Cux-2 protein was undetectable in allregions of the Cux-2�/� P21 mutant telencephalon, including the upper layers of the cortex (left, lower panels). Cux-1 protein expression was indistinguishable between WT andCux-2�/� homozygous P21 mutant mice (right). Bars represent 500 lm (upper panels) and 100 lm (lower panels). (e) Expression of layer-specific markers in the telencephalonof P0 WT and Cux-2�/� mice. Bars represent 100 lm.

Cerebral Cortex August 2008, V 18 N 8 1761

(Fig. 2a). Examination of histological sections showed that both

cell density and the thickness of upper cortical layers (II--IV) of

Cux-2–/– mice was greater than in WT animals (Fig. 2b,c). The

increased cell density was significant in layers II--III, (20%

increment) but was most pronounced in layer IV (30%

increment). In contrast, we observed no differences in

neuronal density in layers V and VI (Fig. 2b). The increased

cell density in the Cux-2–/– upper layers was accompanied by

a moderate increase in the thickness of upper layers (15%

increment) (Fig. 2c). Again, this increment was not observed in

lower layers and resulted in a modest increase in the overall

cortical thickness (Fig. 2c). The combination of increased

cellular density and increased upper layer cortical thickness

indicates an important increase in the total number of neurons

in layers II--III and IV of the Cux-2–/– cortex.

BrdU birthdating experiments performed at E13.5, E14.5,

and E16.5 (Fig. 3) confirmed the selective increase in the

number of upper layer neurons. In accordance with the correct

expression of layer-specific markers (Fig. 1e), the BrdU

experiments suggested normal neuronal migration: neurons

born at E14.5 and E16.5 populated the upper part of the

postnatal cortex (P21) and not the deep cortex in control and

Cux-2–/– animals (Fig. 3a--c). Likewise, neurons born at E13.5

populated the deep cortical layers in both groups of mice (Fig.

3d). However, the number of BrdU-positive neurons that

populated the superficial bins of Cux-2–/– animals and that

were born at E14.5 and E16.5 was increased 40% and 30%,

respectively, in Cux-2–/– mice compared with control mice

(WT or Cux-2+/–) (Fig. 3b,c; bins 2--4 at E16.5 and bins 2--5 at

E14.5). In contrasts, no differences were found between the

populations born at E13.5 (Fig. 3c). Interestingly, the increased

number of labeled upper layer neurons was produced by an

increase in the number of dimly fluorescent BrdU--labeled cells

(Fig. 3a--c, right panels), suggesting that the pulse label had

been diluted by additional rounds of cell division. Hence, upper

layer precursors appear to undergo additional rounds of

division when Cux-2 is removed. Also supporting an increase

in proliferation, the rates of apoptosis in the brains of WT and

Cux-2–/– mice at P0 and P16 postnatal stages were the same

(Fig. 4a,b). Thus, the increment in the number of upper layer

neurons in the Cux-2–/– mice cannot be attributed to defective

neuronal apoptosis. Decreased postnatal apoptosis in Cux-2–/–

mice would also predict less significant differences in the

number of BrdU-labeled upper layer neurons at early postnatal

stages than in the late postnatal days. But we found increased

numbers of E16.5 BrdU--labeled neuronal population in the

Cux-2–/– cortex already at the first postnatal days (P0--P5), and

the increment was equivalent to that found at P21 (Fig. 4c and

see also Fig. 6). All together, we conclude that the increase

number of upper layer neurons in the Cux-2–/– mutant mice is

due to increased proliferation of neuronal precursors during

development and not to decreased apoptosis.

Neuronal Proliferation Is Increased in the SVZ of Cux-2Mutant Mice

The restricted expression of Cux-2 mRNA (Nieto et al. 2004)

and protein in the SVZ (Supplementary Fig. 2) and the increased

upper layer neurons in Cux-2–/– mice suggest that Cux-2 has

specific functions in the proliferation of intermediate precursors

for upper layer neurons. To identify possible differences in cell

cycle parameters, we combined immunostaining for Ki67

antigen, a protein expressed in all dividing cells, and a pulse of

Figure 2. Cell density and thickness of cortical layers in the adult WT and Cux-2�/�cortex. (a) Brains of WT and Cux-2�/� animals (upper panel). Nissl staining ofcortices from WT and Cux-2�/� adult mice. Bar represents 100 lm. (b) Cell densityin each cortical layer of Cux-2�/� and WT mice. (c) Average thickness of the totalcortex and of each layer in the Cux-2�/� adult animals relative to measures taken inthe adult WT cortex. *P\ 0.05, **P\ 0.01 between WT and mutant cortex.

1762 Cux-2 Controls Upper Layer Precursor Proliferation d Cubelos et al.

BrdU to label cells in the S phase. After a 2-h pulse of BrdU in

E15.5 embryos, precursor cells in the SVZ were identified as

those set apart from the BrdU-positive precursors aligned along

the VZ (Fig. 5a). Remarkably, the number of Ki67- and BrdU-

positive cells was dramatically increased in the SVZ of Cux-2–/–

cortex (Fig. 5a, left panel graph). No significant differences were

observed in the proportion of cycling cells that were in the S

phase (i.e., the ratio of BrdU+/Ki67+) between the control and

Cux-2–/– mutant mice. Because this ratio provides an estimate of

cell cycle length, this therefore indicates that Cux-2–/– and

control SVZ precursors cycled at similar rates (Fig. 5a, right

panel graph).

We next studied the number and location of cells undergoing

mitosis with an antibody against the phosphorylated form of

histone 3 (pH3). pH3 staining of E15.5 cortical sections revealed

2 mitotic zones: apical mitosis occurring in radial glial cells at

the ventricular walls and basal mitosis, clearly separated from

the ventricle, occurring in cycling cells of the SVZ (Fig. 5b).

Quantification analysis revealed that the number of cells in basal

mitosis was greatly increased in the SVZ of E15.5 Cux-2–/– mice

compared with their WT counterparts (Fig. 5b, graph). In

contrast, no differences were observed in numbers of cells

undergoing apical mitosis (Fig. 5b, graph). This indicates that

proliferation of VZ precursors, which do not express Cux-2, is

not affected in Cux-2–/– mice and demonstrates a selective

increase in the proliferation of SVZ neuronal precursors but not

of radial glial cells. In agreement with this, the number of Tbr-2

positive cells, which mark intermediate neuronal progenitors in

the proliferative regions (Englund et al. 2005), was dramatically

increased in the cortex of Cux-2–/– mice (Fig. 5c). Similarly,

Figure 3. BrdU birthdating experiments of Cux-2�/� cortical neurons. (a) Representative micrographs of cortical sections taken at P21 showing neurons labeled by BrdUinjection at E16.5 (upper panels) and E14.5 (lower panels) in control (Cux-2þ/?) and Cux-2-deficient (Cux-2�/�) mice. Bar represents 100 lm. The division of the cortex intoequal-sized bins is shown on the right. Inset shows representative examples of bright and dim BrdU-stained nuclei. Quantitative analysis of the distribution of BrdU populationslabeled at E16.5 (b), E14.5 (c), and E13.5 (d) and examined at P21. Black and gray bars represent bright and dim BrdU-stained nuclei, respectively. The left-hand charts show thedistribution of BrdU-labeled cells in each of 10 equal bins dividing the cortex. *P\ 0.01 as analyzed by a 10 3 2 chi-square matrix test. The upper right-hand charts show thetotal number of cells labeled in all sections of control and Cux-2�/� mice. Lower right charts show the relative proportion of dark and dim BrdU-stained nuclei. *P\ 0.01 asanalyzed by 2-tailed Student’s t-test.

Cerebral Cortex August 2008, V 18 N 8 1763

analysis of the number of Tbr-2 and pH3 in the SVZ of E14.5

revealed increments of proliferating SVZ cells in E14.5 Cux-2–/–

mice compared with WT animals. In contrast, no differences

were found on cortical basal mitosis at E12.5, when lower layer

neurons are generated (Supplementary Fig. 3).

Cux-2 Regulates the Number of Upper Cortical NeuronsIndependently of Cux-1

VZ and SVZ cells express low levels of Cux-1 protein, which

also shows persistent expression in upper layer neurons (Nieto

et al. 2004). To investigate the possibility that Cux-1 and Cux-2

might show redundant or interacting roles, we examined Cux-

1–/–; Cux-2–/– double-mutant mice. This double gene deletion

is embryonically lethal for reasons that are still undetermined.

The lethality of Cux-1–/–; Cux-2–/– double mutation is due to

defects manifest at early embryonic stages (between E9 and

E15) (Cubelos B and Nieto M., unpublished data). However,

although this phenotype is highly penetrant, a small proportion

of mice develop to birth. Nissl staining of P0 coronal sections

showed no gross abnormalities in the double and single Cux-1

and Cux-2 mutants and their cortex appeared to be normally

laminated (Fig. 6a). We analyzed the migration and the number

of E16.5 BrdU-labeled neurons at birth (P0) in Cux-1–/–; Cux-

2–/– double mutants, compared with single Cux-2-/- mutants,

and control littermates (Fig. 6b,c). The migration patterns of

BrdU-labeled cells to the upper layer of the cortex were

indistinguishable in all genotypes. However, the number of

E16.5-birthdated neurons was significantly increased in the

cortex of Cux-1–/–; Cux-2–/– double-mutant mice compared

with controls, but this increase was no greater than that

observed in the Cux-2 single-mutant mice (Fig. 6c). These

experiments rule out the possibility that an unrepressed

function of Cux-1 is responsible for the increased proliferation

of upper layer neurons in Cux-2 mutant mice. They moreover

provide evidence that the control of upper layer neuron

number is a unique function of Cux-2 and is not redundant

with Cux-1 activities.

Cux-2 Controls Reentry of Precursor Cells into the CellCycle in a Cell-Autonomous Manner

The higher number of weakly BrdU-labeled cells in the cortex

of Cux-2–/– mice suggested that neuronal precursors in these

animals underwent more cell divisions than WT cells immedi-

ately before the generation of upper layer neurons. To

investigate this further, we set out to unequivocally identify

whether cortical neurons had been generated from precursors

that had divided once, or more than once, by sequential

Figure 3. Continued.

1764 Cux-2 Controls Upper Layer Precursor Proliferation d Cubelos et al.

Figure 4. Brains of early postnatal Cux-2�/� mice contain an excess of upper cortical neurons that is not caused by decreased apoptosis. (a) Detection of apoptotic cells withantiactive caspase-3 antibody on cortical sections of P0 control and Cux-2�/� animals. Inset shows detail of caspase-3 positive cell. (b) Quantification of antiactive caspase-3staining of cortical sections of P0 (left) and P16 (right) control and Cux-2�/� animals; no differences were detected at either stage. (c) E16.5-birthdated (BrdU-labeled) neuronsin the P5 cortex of control and Cux-2�/� littermates. Bars represent 100 lm (a) and 25 lm (c).

Cerebral Cortex August 2008, V 18 N 8 1765

Figure 5. Proliferation is increased in the SVZ of Cux-2�/� mutant cortex. (a) Staining of Ki67, a protein expressed in all dividing cells (green), and BrdU-labeled dividing cells inS phase (red) after a 2-h pulse of the nucleotide in E15.5 cortical sections. The left-hand bar chart shows quantification of Ki67-positive and BrdU-positive cells (n 5 3). The right-hand chart shows the proportion of dividing cells in S phase (BrdU positive) (number of BrdUþ/number of Ki67þ) in control and Cux-2�/� littermates (n 5 3). (b) Detection ofcortical pH3-positive cells undergoing mitosis at E15.5. The chart shows the relative numbers of mitotic cells in the VZ and SVZ of Cux-2�/� and control littermates, with thenumber of cells in the control mice set at 100%. Bar represents 50 lm (a and b). (c) Immunodetection of Tbr-2 positive cells in the SVZ of E15.5 cortical sections.

1766 Cux-2 Controls Upper Layer Precursor Proliferation d Cubelos et al.

injection with 2 different halogenated nucleotide analog. The

nucleotide analog IdU was given in a first injection, and CldU

was injected 10 h later. The interval between injections allows

many cells to exit the S phase after the first nucleotide

injection but is shorter than the whole cell cycle, ensuring that

cells reentering the cell cycle are labeled with the second

nucleotide. Thus, cells that exited the cell cycle after the first

injection are labeled with IdU only, whereas cells generated

after subsequent subdivisions are double labeled with IdU and

CldU (see diagram Fig. 7c). Cells that were not in the S phase at

the first injection, or that diluted any incorporated IdU

sufficiently during successive divisions, appear labeled only

with CldU. We analyzed the upper layer neurons labeled at

E14.5 in postnatal (P21) brain sections of control and Cux-2–/–

animals (Fig. 7), and we found that the number of double-

positive cells was much greater in the Cux-2–/– cortex (Fig.

7a). The ratio between the number of CldU-positive cells

(second injection; red cells) and the total number of IdU-

postive cells (first injection; green cells) shows that the

proportion of precursor cells that reentered the cell cycle

after the first injection was 2.5-fold higher in the Cux-2–/– mice

than in control animals (Fig. 7b). Thus, our double-labeling

analysis suggests that Cux-2 controls exit from the cell cycle

(Fig. 7c,d).

To investigate whether the phenotype of Cux-2-deficient

precursors reflects a cell- or noncell-autonomous function, we

overexpressed Cux-2 in dividing embryonic neural stem cells

grown as neurospheres in vitro. After cotransfection with Cux-2,

or the empty vector (control), together with a GFP reporter,

dissociated single GFP-positive cells were sorted by flow

cytometry fluorescence activated cell sorting (FACS) (Fig. 7e).

Sorted cells were then seeded at clonal density, and the number

of neurospheres was quantified after 7 days in culture. As shown

in Figure 7f,g, overexpression of Cux-2 significantly reduced the

number of neurospheres generated from the sorted cells,

compared with control cells. Given that Cux-2-expressing cells

and control cells were equally able to survive and differentiate

into glia and neurons when plated in differentiating conditions

(not shown), the reduced neurosphere formation by Cux-2

overexpressing progenitors was not due to increased death rate

or to changes in cell fate. These results therefore strongly

suggest that Cux-2 is able to direct cells out of the cell cycle in

a cell-autonomous manner.

Discussion

In the present study, we demonstrate that expression of Cux-2

selectively controls the proliferation of intermediate neuronal

precursors in the SVZ and the number of upper layer neurons.

In the context of evolution, the actions of Cux-2 during

development might provide mechanisms controlling the

appearance and expansion of upper layer neurons.

Our data indicate that the restricted expression of Cux-2 in

the SVZ selectively controls the proliferation rates of neuronal

intermediate precursors during mouse development. The role

of Cux-2 in cell cycle regulation was previously suggested by

Figure 6. Analysis of the E16.5-birthdated neuronal population in Cux-1; Cux-2 double-mutant mice. (a) Nissl staining of coronal sections of the telencephalon of control(Cux-2þ/?), Cux-2�/�, and Cux-1; Cux-2 double-mutant mice at P0. (b) Micrographs show staining of E16.5-birthdated (BrdU stained) neurons in sections of the P0 cortex ofcontrol and Cux-1; Cux-2 double-mutant mice. (c) The chart shows the percentage (relative to control animals) of E16.5-birthdated neurons in the P0 cortex of control, Cux-1;Cux-2 double mutant, and Cux-2�/� animals. Bars represent 500 lm (a) and 100 lm (b).

Cerebral Cortex August 2008, V 18 N 8 1767

its reported expression in tissues undergoing extended pro-

liferation during embryonic development such as the limb bud

progress zones or the urogenital system (Iulianella et al. 2003)

but has not been demonstrated before. Hence, we herein

provide the first evidence demonstrating a role for Cux-2 in

cell proliferation.

Interestingly, our studies demonstrate that Cux-2 limits the

number of times SVZ neuronal precursors go through the cell

cycle. Our neuronal birthdating and double-labeling experi-

ments demonstrate that Cux-2–/– upper layer precursors

reenter the cell cycle at a higher frequency than their WT

counterparts (Fig. 7d). Concurrently, we observed an expan-

sion of intermediate neuronal precursors in the SVZ of the

developing Cux-2–/– mice. This selective expansion is most

definitively demonstrated by an increase in Tbr-2-expressing

cells but no changes in precursors of the VZ. This selective

Figure 7. Cux-2 regulates cell cycle exit. (a) Confocal images of neurons stained for IdU (green) and CldU (red) in cortical sections of the brains of P21 WT and Cux-2�/� micethat had been sequentially injected with the nucleotides at E14.5 (IdU, first injection; CldU, second injection: 10-h interval). Arrowheads mark single-labeled IdU-positive cells.Arrows mark single-labeled CldU-positive cells. Double-labeled cells (yellow on merged panel, double arrows) identify neurons generated from precursor cells that underwent 2 ormore rounds of division after the initial injection with IdU. Bar represents 50 lm. (b) Double-positive cells (IdUþ and CldUþ) as a percentage of the total number of IdUþ cells incontrol and Cux-2-deficient mice, providing an estimate of the proportion of neurons generated from precursors that reentered the cell cycle after the initial IdU injection.*P\ 0.01. (c) Schematic representation of the experimental design. (d) Cux-2 may promote reentry of SVZ precursors into the cell cycle (N, neuron). The cell with red nucleusrepresents proliferating SVZ precursors. (e) Flow cytometry histogram of cells nucleotransfected with Cux-2 and a GFP reporter. (f) Micrographs showing neurospheres after 10days in culture by GFP-positive cells cotransfected with a DNA construct expressing Cux-2 or the empty vector. Bar represents 100 lm. (g) Neurospheres formation asa proportion of total cells plated. *P\ 0.01 (n 5 3).

1768 Cux-2 Controls Upper Layer Precursor Proliferation d Cubelos et al.

effect of Cux-2 in SVZ cells supports the notion that the

functions of Cux-2 are cell autonomous. Cell-autonomous

mechanisms are also strongly supported by our experiments

showing that overexpression of Cux-2 decreases proliferation

in neural progenitor cells in vitro.

The increased numbers in the number of SVZ precursors in

Cux-2–/– mice are quite significant (about 40% increment).

This excessive proliferation temporally correlates with in-

creased production of late-born neurons that migrate to the

upper layers in normal patterns (30--40% in BrdU experiments).

This causes parallel increments in cell density of layers II--IV

(20--30% increment), as well as a moderate increase in upper

layer thickness (15% increment).

Somewhat surprisingly, Cux-2 does not appear to be

functionally redundant with Cux-1 in the SVZ precursor. The

nonredundancy of Cux-2 in intermediate neuronal precursors

is suggested by its restricted expression in the SVZ, which is

not shown by Cux-1, because it is expressed in VZ precursors

as well. Our analysis of Cux-1–/–; Cux-2–/– double-mutant mice

confirms this notion and demonstrates that the proliferation of

upper layer neuronal precursor does not depend on Cux-1

activity. The 2 Cux genes thus do not cooperate to control

these aspects of neuronal development.

Our findings are of particular interest in the search for

a mechanism to explain how the appearance and selective

expansion of upper cortical layers during development evolved

(Marin Padilla 2001; Hill and Walsh 2005; Kriegstein et al. 2006;

Martinez-Cerdeno et al. 2006). A larger cortex implies that

precursor cells undergo more rounds of division. In addition,

the extended period of neurogenesis adds the upper layer to

the cortex. Indeed, upper cortical layer neurons are highly

represented in the primate cerebral cortex, especially in

humans (reviewed in Hill and Walsh 2005). During develop-

ment and evolution, inhibition of Cux-2-mediated cell cycle

exit in the SVZ could be a target mechanism to expand the

number of upper layer neurons without affecting the number

of deep layer neurons. Thus, the functions of Cux-2 fulfill both

of the developmental and evolutionary necessities of expand-

ing the number of layers. In this regard, evolution might have

required Cux-2 functions in processes such as the addition of

upper layer neurons to the 3 layers of the reptilian cortex, the

later selective expansion of upper layers in higher mammals

(Reiner 1991; Marin Padilla 2001; Kriegstein et al. 2006), or the

focal expansion of the upper layers and the SVZ that has been

proposed to occur during the formation of the gyrencephalic

cortex (Kriegstein et al. 2006).

Supplementary Material

Supplementary figures 1--3 can be found at: http://www.cercor.

oxfordjournals.org/.

Funding

Ministerio de Educacion y Ciencia (SAF2005--0094); Mutua

Madrilena Automovilistica (2004); RYC-2003-006143 to M.N.;

JdC-05-162-74c to B.C.; the Ministerio de Educacion y Ciencia

(BES-2006-13901) to A.S.-S.; National Institute of Neurological

Disorders and Stroke (2RO1 NS032457) to C.A.W.; the Spanish

Ministry of Health and Consumer Affairs and the Pro-

CNIC Foundation to Centro Nacional de Investigaciones

Cardiovasculares.

Notes

We thank B. Alarcon, S. Bartlett, F. Guillemot, M. Mellado, J. M.

Rodrıguez-Frade, P. Bovolenta, M. Guzman, and H. M. van Santen for

critical reading of the manuscript and for their experimental advice.

We thank A. Nepveu (McGill University, Canada) for the anti-Cux-2

antibody, M. Sheng (Picower Institute, MIT, Cambridge, MA) for the

Tbr-1 antiserum, and A. J. van Wijnen (University of Massachusetts

Medical School, Worcester, MA) for the Cux-1 mutant mice. We are

grateful to S. Montalban, S. Gutierrez-Erlandsson, and D. Esteban for

technical assistance. During the generation of theCux-2loxP conditional

mice,M.N. was a Lefler fellow in the laboratory of C.A.W.We are indebted

to C.A.W. for allowing us to continue the project at the Centro Nacional

de Biotecnologıa (Madrid, Spain). C.A.W. is an Investigator at the

Howard Hughes Medical Institute. Conflict of Interest : None declared.

Address correspondence to Email: mnlopez@cnb.uam.es.

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